It is the aim of this research proposal to dissect the requirements for the in vitro generation of suppressor T cells (Ts). As suppressor cells have been implicated in the normal maintenance of immune system homeostasis, a better understanding of the complex requirements needed to generate these important regulatory cell populations will hopefully enable us to better comprehend and rectify Ts defects in autoimmune diseases, immunodeficiency disorders, and cancer. To this end we have developed an in vitro system for the generation of 2nd order (Ts2) and effector suppressor cells (Ts3) specific for the 4-hydroxy-3-nitrophenyl acetyl hapten (NP). Cyclophosphamide treated responder populations, which are functionally depleted of both Ts2 and Ts3 cells, are challenged in vitro with the T dependent antigen NP-horse red blood cells (NP-HRBC) for 5 days. On the 4th day of culture, separate subcultures containing experimental Ts2 and Ts3 populations are added to responder cultures. One day later, the direct anti-NP specific plaque forming cell (PFC) responses are determined. This methodology will allow us to directly identify and fully characterize both precursor and mature Ts populations, the role of accessory cells, soluble mediators, and the genetic restrictions which control the differentiation and expression of Ts activity. Phenotypic analysis of both mature and precursor Ts populations with a panel of monoclonal reagents may allow us to identify markers that are differentially expressed on various Ts subsets and to determine when Ts develop their idiotypic specificities. By analyzing the ability of fetal liver and thymic cell populations to generate Ts2 or Ts3 activity, we will be in a position to determine when suppressor cells become immunocompetent. We will also examine the role of accessory cells in the generation of Ts. We will perform experiments to determine whether MHC homology between the macrophages and T or B cells is required for suppressor cell induction and whether factors from macrophages influence the induction process. In addition the role of B cells in Ts induction would be examined and the general question of the importance of B cell receptors in the development of T cells can be addressed. Finally a better understanding of the requirements for suppressor cell induction may enable us to develop a method for maintaining long-term, untransformed suppressor T cell lines in culture.